Young people (15-24 years old [yo]) acquire half of the 20 million new sexually transmitted infections (STI) annually in the U.S. and 1 in 4 sexually active adolescent girls has an STI. Complex biological, socio- behavioral, and cultural factors place sexually active adolescent girls at higher risk of acquiring STIs compared with boys and adult women. Some STIs can increase HIV acquisition by breaching the protective mucosal epithelial barrier, promoting inflammation, and recruiting HIV target cells into the genital tract. This synergy between STIs, inflammation, and HIV contributes to the 380,000 new HIV infections among adolescent girls and young women (10-24yo) each year worldwide. In communities where STI and HIV prevalence are high, sexually active girls using hormonal contraception (HC), but without barrier protection, are at risk of STI/HIV acquisition. Several observational studies have suggested that depot medroxyprogesterone acetate (DMPA) may increase the risk of HIV acquisition by up to 3.0-fold. Yet, the biological aspects of STI co-infections, inflammation, exogenous hormones, and HIV acquisition in adolescent girls and young women are understudied. Given that most HIV infections occur at mucosal surfaces, there is a critical need to better understand mucosal immune function and biologic factors like hormonal status and vaginal microbiota that can alter susceptibility to STI/HIV among adolescent girls and young women. In the absence of such knowledge, the development of effective biomedical technologies to prevent STIs and HIV within this vulnerable key population will likely remain difficult. Our central hypothesis is that the hypoestrogenemia induced by DMPA decreases the vagina's natural host defense mechanisms against STI/HIV by altering the microbiota (e.g. decreasing lactobacilli), decreasing mucus pathogen trapping properties, and increasing inflammation. To test our hypothesis, we propose the following two specific aims: (1) To identify the association between the vaginal microbiota, inflammation, and STIs. The vaginal microbiota of 225 menarcheal, sexually-active, healthy U.S. adolescent girls and young women (13-24yo) will be characterized by 16s rRNA sequencing using self- collected vaginal swabs in a cross-sectional design; and (2) To determine the impact of DMPA use on vaginal microbiota, inflammation, and female genital tract anatomy and physiology. A subset of adolescent girls and young women who initiate DMPA (n=40) or those not using any HC (n=40) will be followed prospectively. Changes from baseline in the vaginal microbiota, inflammation, and cervicovaginal mucus properties will be assessed at 3 and 12 weeks. Our approach is innovative because it seeks to address key issues in an understudied population using cutting edge methods. The proposed research is significant because it is expected to vertically advance the field by providing key insight into the important role of vaginal microbiota and exogenous hormones, which may lead to a new approach to HIV prevention among adolescent girls and young women.